Eker rats are susceptible to spontaneous and carcinogen-induced renal cell carcinoma (RCC) due to a germline defect in the tuberous sclerosis 2 (TSC-2) tumor suppressor gene. These animals serve as useful models for studying mechanisms of renal carcinogenesis and the role of the TSC-2 gene in this process. While it is well established that loss of TSC-2 gene function contributes to tumor development in rodents and humans, the normal function(s) of tuberin, the product of the TSC-2 gene, and the mechanism by which loss of function contributes to tumorigenesis are not clear. Recently, data have emerged implicating tuberin as a negative regulator of signaling downstream of AKT. In this renewal application, we will test the hypothesis that tuberin is itself a target for AKT phosphorylation, and that this phosphorylation in conjunction with a PDZ binding domain at tuberin's carboxy terminus regulates tuberin's binding partners, subcellular localization and function within the cell. In Specific Aim 1 we will test the hypothesis that tuberin is a target for AKT phosphorylation. In Specific Aim 2 we will identify tuberin binding partners with the potential to exercise spatial control of tuberin function. In Specific Aim 3 we will determine the functional consequences of tuberin interaction with 14- 3-3 or its PDZ binding partner nNOS on cell signaling downstream of AKT, RHO-mediated cell adhesion and nNOS activity. Studies such as these on the normal function of tuberin have the potential to reveal new interactions between tuberin and cell signaling pathways that can yield valuable information regarding how the TSC-2 suppressor gene participates in renal carcinogenesis.